CN109929871B - Method for mediating double-stranded RNA to enter Chinese purple beetle body - Google Patents
Method for mediating double-stranded RNA to enter Chinese purple beetle body Download PDFInfo
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Abstract
The invention relates to a method for mediating double-stranded RNA to enter into the body of Chinese purple beetle, belonging to the technical field of genetic engineering. The method comprises the steps of firstly extracting total RNA of Chinese purple beetles, carrying out reverse transcription to cDNA, carrying out PCR amplification, carrying out double enzyme digestion on an amplification product and an L4440 vector by SacI and SalI, connecting enzyme digestion products by T4 ligase overnight, transferring the obtained FAD-L4440 recombinant plasmid into HT115 competent cells, and carrying out amplification culture until bacterial liquid OD is obtained 600 And (3) adding IPTG to induce for 2-5 h to obtain FAD-L4440-HT115 thalli, diluting the thalli, and directly smearing the thalli on larva stage insects of the Chinese red beetles. The method can transfect dsRNA into the lac insect body, effectively interfere the FAD gene expression quantity, thereby reducing the glue secretion quantity of individuals, and providing reference for insects which are not suitable for transfection methods such as injection, feeding and the like in the RNAi transfection process.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a method for mediating double-stranded RNA to enter a Chinese purple beetle body.
Background
Chinese purple beetleKerria chinensis) Belonging to Hemiptera, gecko (Tachardii) and Gecko genusKerria) Is a resource insect with important economic value, mainly is mainly registered in the Dalbergia odoriferaDalbergia cocrinchinensis) Radix AlbiziaeAibizia lucida) On the plants, the plant phloem juice is sucked for growth and reproduction. Shellac is a purely natural resin secreted by females through glands, and the main components of the shellac are shellac resin, shellac wax, shellac acid and the like. The shellac has the excellent characteristics of strong adhesion, good insulativity, moisture resistance, smooth coating film and the like, and has the excellent physical and chemical properties of no toxicity, no smell and the like, so that the shellac is widely applied to the industries of military industry, daily chemical industry, electronics and the like, and has important economic value.
RNA interference (RNAi) refers to the phenomenon in which double-stranded RNA (double-strandedRNA, dsRNA) induces homologous mRNA degradation in eukaryotes, silencing target gene expression. dsRNA transfection includes injection, feeding, soaking, virus infection, transgene and other methods, and injection and feeding are more common. In the initial stage of RNA interference research, injection is mainly applied to nematodes initially, and along with development of application in drosophila, plutella xylostella is currently usedPlutella xyllostella) Brown planthopper and its production processNilaparvata lugens) Radix Et rhizoma Rhei and radix Et rhizoma ValerianaeTribolium castaneum) The system for inducing RNAi by injecting dsRNA is established in the study subjects. However, when the individual study object is too small, the problem of increased injection difficulty and reduced survival rate cannot be effectively solved. The feeding method for inducing RNAi has the characteristics of simple and convenient operation, small harm to the researched objects and the like, and is characterized by potato beetlesLeptinotarsa decemlineata) Chinese patent drugSitobion avenae) And the like. However, the feeding method has the defects of slower action and lower efficiency. Because of the disadvantages of injection and feeding methods, it is important to use which transfection method to transfect dsRNA into insects when the subject individual is too small to feed.
The insect species of the study, namely the purple beetle, is smaller in individual, is a piercing-sucking mouth gag, and can grow and reproduce by sucking the juice of the plant after the mouth needle pierces the host plant for life. Thus, which transfection mode is selected by lac is critical to verifying the function of the relevant gene using RNAi. Therefore, how to overcome the defects of the prior art is an urgent problem to be solved in the technical field of the current genetic engineering.
Disclosure of Invention
The invention aims to solve the defects of the prior art, and provides a method for mediating double-stranded RNA to enter the body of Chinese red beetles, which can transfect dsRNA into the body of Chinese red beetles and provides references for insects which are not suitable for transfection methods such as injection, feeding and the like in the RNAi transfection process.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a method for mediating double-stranded RNA into the body of rhodochrous sinensis, comprising the steps of:
step (1), extracting total RNA of the Chinese red beetles, carrying out reverse transcription to cDNA, and then carrying out PCR amplification, wherein an amplification product and an L4440 vector are subjected to double enzyme digestion by SacI and SalI, and the enzyme digestion product is connected overnight by using T4 ligase to obtain FAD-L4440 recombinant plasmid;
step (2), transferring the FAD-L4440 recombinant plasmid obtained in the step (1) into HT115 competent cells, and performing amplification culture until bacterial liquid OD is obtained 600 Adding IPTG to induce for 2-5 h to obtain FAD-L4440-HT115 bacteria at 0.1-0.7;
step (3), diluting FAD-L4440-HT115 cells obtained in the step (2) to a concentration of 6.2ng -1 ~620ng•mL -1 The diluted bacterial liquid is directly smeared on the larva stage insect body of the Chinese red beetle.
Further, it is preferable that the total RNA of chinese purple beetle comprises total RNA of chinese purple beetle FAD gene.
Further, it is preferable that in the step (1), the primers used for the PCR amplification are ds-F and ds-R, wherein,
ds-F：atgagctcgcaatgatacaacgaacca；
ds-R：atgtcgacgaacgatgtgaccataagc。
further, it is preferable that in the step (1), the PCR amplification system is
Taq PCR Master Mix(2X, with Red Dye) 12.5μl，
cDNA 1.0μl，
ds-F 10μM 1.0μl，
ds-R 10μM 1.0μl，
dH2O 9.5μl，
And a total of 25.0 μl.
Further, it is preferable that in the step (1), the PCR reaction conditions are as follows: pre-denaturation at 95℃for 2min; denaturation at 95℃for 30s, annealing at 50℃for 30s, elongation at 72℃for 1min,35 cycles; extending at 72deg.C for 10min, and preserving at 4deg.C.
Further, it is preferable that in the step (1), the time for the cleavage is 10 minutes and the temperature is 37 ℃.
Further, it is preferable that in the step (1), the system of cleavage is:
SacⅠ 1.0 μl，
SalⅠ 1.0μl，
Buffer 5.0 μl，
FAD or L4440.0. Mu.l,
dH2O 38.0 μl，
and total 50.0 μl.
Further, preferably, in the step (2), the specific method of the expansion culture is as follows: the constructed FAD-L4440 recombinant plasmid is transformed into HT115 competent strain, coated on SOC solid culture medium containing ampicillin and tetracycline, cultured for 12h, single colony is selected and transferred into SOC liquid culture medium containing 100 mug/mL ampicillin and 50 mug/mL tetracycline for overnight culture at 37 ℃, then the overnight culture solution is added into 2 XYT liquid culture medium containing 75 mug/mL ampicillin and 12.5 mug/mL tetracycline for culturing to OD 600 =0.1~0.7。
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, an RNAi system for expressing dsRNA by bacteria is introduced in the gene function verification of the purple beetle for the first time, and comparison of two treatment methods of coating and spraying shows that the coating method can effectively interfere the FAD gene expression quantity, so that the individual glue yield is reduced, but the interference effect of the spraying method is not obvious. The existing dsRNA transfection method mainly uses an injection method and a feeding method, but when an individual of a study object is too small, the problems of increased injection difficulty and reduced survival rate still cannot be effectively solved, the feeding method also has the defects of slower action and lower efficiency, and when the individual of the study object is too small and cannot feed, the coating method adopted by the study can provide references for insects which are not suitable for the transfection methods such as the injection method, the feeding method and the like in the RNAi transfection process, and an effective technical method is provided for verifying functions of genes related to lac synthesis on a molecular level.
The invention adopts a coating method, and discovers that the low-concentration and medium-concentration bacterial liquid transfection has obvious interference effect on the FAD gene of the rhodococcus neoformans, which is respectively reduced by 90.79 percent and 85.46 percent, and the gum secretion of individuals is obviously different from that of ck groups, which is respectively reduced by 14.89 percent and 12.73 percent. Therefore, the fact that the FAD gene expression quantity can be effectively interfered by a coating method is verified, and the glue yield of an individual is reduced.
Drawings
FIG. 1 is a flow chart of construction of recombinant vector and dsRNA induced expression;
FIG. 2 is an electrophoretic pattern of interference vector construction and dsRNA-induced expression; wherein, M, DNA Marker;1, FAD gene fragment PCR product; 2, FAD-L4440 plasmid amplified by ds-F and ds-R; 3, FAD-L4440 PCR product amplified by T7 single primer; 4, FAD-L4440-HT115 bacterial liquid is subjected to RNA electrophoresis before induction; 5, FAD-L4440-HT115 bacterial liquid induced RNA electrophoresis.
FIG. 3 is a graph showing the effect of bacterial liquid coating at different concentrations on the Hua Zijiao FAD gene; a is the gene expression level after the treatment of the low-concentration bacterial liquid; b is the gene expression level after the treatment of the medium concentration bacterial liquid; c is the gene expression level after the treatment of the high-concentration bacterial liquid. Capital letters indicate differences in gene expression levels between two bacterial fluid treatments during the same time periodAnalysis of the differenceP< 0.05); lowercase letters indicate the analysis of the difference of gene expression levels at different time periods under the same treatmentP＜0.05）。
FIG. 4 is a graph showing the effect of spraying Shi Duizhong Hua Zijiao FAD genes on bacterial liquids of different concentrations; a is the gene expression level after the treatment of the low-concentration bacterial liquid; b is the gene expression level after the treatment of the medium concentration bacterial liquid; c is the gene expression level after the treatment of the high-concentration bacterial liquid. Capital letters indicate analysis of the difference between the gene expression levels of two bacterial solutions treated in the same time periodP< 0.05); lowercase letters indicate the analysis of the difference of gene expression levels at different time periods under the same treatmentP＜0.05）。
FIG. 5 is a graph showing the results of the measurement of the amount of gum secreted by individuals after RNA interference.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The materials or equipment used are conventional products available from commercial sources, not identified to the manufacturer.
Example 1
A method for mediating double-stranded RNA into the body of rhodochrous sinensis, comprising the steps of:
step (1), extracting total RNA of the Chinese red beetles, carrying out reverse transcription to cDNA, and then carrying out PCR amplification, wherein an amplification product and an L4440 vector are subjected to double enzyme digestion by SacI and SalI, and the enzyme digestion product is connected overnight by using T4 ligase to obtain FAD-L4440 recombinant plasmid;
step (2), transferring the FAD-L4440 recombinant plasmid obtained in the step (1) into HT115 competent cells, and performing amplification culture until bacterial liquid OD is obtained 600 Adding IPTG to induce for 2h to obtain FAD-L4440-HT115 thallus with the volume of 0.1;
step (3), diluting FAD-L4440-HT115 cells obtained in the step (2) to a concentration of 6.2ng -1 The diluted bacterial liquid is directly smeared in the middle partOn the larvae of the shellac.
Example 2
A method for mediating double-stranded RNA into the body of rhodochrous sinensis, comprising the steps of:
step (1), extracting total RNA of the Chinese red beetles, carrying out reverse transcription to cDNA, and then carrying out PCR amplification, wherein an amplification product and an L4440 vector are subjected to double enzyme digestion by SacI and SalI, and the enzyme digestion product is connected overnight by using T4 ligase to obtain FAD-L4440 recombinant plasmid;
step (2), transferring the FAD-L4440 recombinant plasmid obtained in the step (1) into HT115 competent cells, and performing amplification culture until bacterial liquid OD is obtained 600 Adding IPTG to induce for 5h to obtain FAD-L4440-HT115 thallus at 0.7;
step (3), diluting FAD-L4440-HT115 cells obtained in the step (2) to a concentration of 620ng -1 The diluted bacterial liquid is directly smeared on the larva stage insect body of the Chinese red beetle.
In the step (1), the total RNA of the Chinese violet worm comprises the total RNA of the FAD gene of the Chinese violet worm.
The PCR amplification adopts the primers of ds-F and ds-R, wherein,
ds-F：atgagctcgcaatgatacaacgaacca；
ds-R：atgtcgacgaacgatgtgaccataagc。
the PCR amplification system is
Taq PCR Master Mix(2X, with Red Dye) 12.5μl，
cDNA 1.0μl，
ds-F 10μM 1.0μl，
ds-R 10μM 1.0μl，
dH2O 9.5μl，
And a total of 25.0 μl.
The PCR reaction conditions are as follows: pre-denaturation at 95℃for 2min; denaturation at 95℃for 30s, annealing at 50℃for 30s, elongation at 72℃for 1min,35 cycles; extending at 72deg.C for 10min, and preserving at 4deg.C.
The time for the cleavage was 10min and the temperature was 37 ℃.
The enzyme digestion system is as follows:
SacⅠ 1.0 μl，
SalⅠ 1.0 μl，
Buffer 5.0 μl，
FAD or L4440.0. Mu.l,
dH2O 38.0 μl，
and total 50.0 μl.
In the step (2), the specific method for the expansion culture comprises the following steps: the constructed FAD-L4440 recombinant plasmid is transformed into HT115 competent strain, coated on SOC solid culture medium containing ampicillin and tetracycline, cultured for 12h, single colony is selected and transferred into SOC liquid culture medium containing 100 mug/mL ampicillin and 50 mug/mL tetracycline for overnight culture at 37 ℃, then the overnight culture solution is added into 2 XYT liquid culture medium containing 75 mug/mL ampicillin and 12.5 mug/mL tetracycline for culturing to OD 600 =0.7。
Example 3
A method for mediating double-stranded RNA into the body of rhodochrous sinensis, comprising the steps of:
step (1), extracting total RNA of the Chinese red beetles, carrying out reverse transcription to cDNA, and then carrying out PCR amplification, wherein an amplification product and an L4440 vector are subjected to double enzyme digestion by SacI and SalI, and the enzyme digestion product is connected overnight by using T4 ligase to obtain FAD-L4440 recombinant plasmid;
step (2), transferring the FAD-L4440 recombinant plasmid obtained in the step (1) into HT115 competent cells, and performing amplification culture until bacterial liquid OD is obtained 600 Adding IPTG to induce for 3h to obtain FAD-L4440-HT115 thallus at 0.5;
step (3), diluting FAD-L4440-HT115 cells obtained in the step (2) to a concentration of 300ng.mL -1 The diluted bacterial liquid is directly smeared on the larva stage insect body of the Chinese red beetle.
In the step (1), the total RNA of the Chinese violet worm comprises the total RNA of the FAD gene of the Chinese violet worm.
The PCR amplification adopts the primers of ds-F and ds-R, wherein,
ds-F：atgagctcgcaatgatacaacgaacca；
ds-R：atgtcgacgaacgatgtgaccataagc。
the PCR amplification system is
Taq PCR Master Mix(2X, with Red Dye) 12.5μl，
cDNA 1.0μl，
ds-F 10μM 1.0μl，
ds-R 10μM 1.0μl，
dH2O 9.5μl，
And a total of 25.0 μl.
The PCR reaction conditions are as follows: pre-denaturation at 95℃for 2min; denaturation at 95℃for 30s, annealing at 50℃for 30s, elongation at 72℃for 1min,35 cycles; extending at 72deg.C for 10min, and preserving at 4deg.C.
The time for the cleavage was 10min and the temperature was 37 ℃.
The enzyme digestion system is as follows:
SacⅠ 1.0 μl，
SalⅠ 1.0 μl，
Buffer 5.0 μl，
FAD or L4440.0. Mu.l,
dH2O 38.0 μl，
and total 50.0 μl.
In the step (2), the specific method for the expansion culture comprises the following steps: the constructed FAD-L4440 recombinant plasmid is transformed into HT115 competent strain, coated on SOC solid culture medium containing ampicillin and tetracycline, cultured for 12. 12h, single colony is selected and transferred into SOC liquid culture medium containing 100 mug/mL ampicillin and 50 mug/mL tetracycline for 37 ℃ overnight culture, then the overnight culture solution is added into the strain containing 75 mug/mL ampicillin and 12.5 mugCulturing in 2 XYT liquid culture medium containing/mL tetracycline to OD 600 =0.5。
Application instance
All media involved in the experiments were purchased from the institute of biotechnology (Shanghai) Co., ltd, wherein:
SOC medium composition: 20.0g of peptone; 5.0g of yeast powder; sodium chloride 0.5g; 5.0g of magnesium sulfate heptahydrate; d-glucose 3.6g.
SOC solid medium composition: SOC medium: 34g, agar powder: 12g, distilled water: 1L
SOC liquid medium composition: SOC medium: 34g, distilled water: 1L
Ampicillin-containing SOC solid/liquid medium: 100. Mu.g of ampicillin per ml of medium was present.
SOC solid/liquid medium containing ampicillin and tetracycline: 100. Mu.g of ampicillin per ml of medium contained 50. Mu.g of tetracycline.
2 XYT medium composition: 10.0g of yeast powder; 16.0g of peptone; 5.0g of sodium chloride.
2 XYT liquid medium containing ampicillin and tetracycline: each milliliter of the culture medium contains 75 mug of ampicillin and 12.5 mug of tetracycline.
Note that: herein "/" means "or".
The invention relates to a method for effectively transfecting double-stranded RNA into an insect body by adopting a coating method to cause the reduction of gum secretion in the verification of the gene function of Chinese lac, and mainly relates to a method for transfecting double-stranded RNA in the verification of the gene function of Chinese lac. The main purpose of the invention is as follows: 1) Exploring a high-efficiency and convenient transfection method for mediating double-stranded RNA into insect bodies; 2) Comparing the coating method with the spraying method, and determining which method is an effective transfection method; 3) Adopting three modes of high, medium and low dsRNA bacterial liquid concentration, which concentration can achieve the best silencing effect; from the three purposes, the efficient transfection of double-stranded RNA into the lac worm body to cause the silence of the expression of the related genes is discussed, and an effective technical method is provided for verifying the functions of the genes related to lac synthesis at the molecular level.
1. Construction of recombinant vectors
Total RNA of Trichinella sinensis was extracted using an RNA extraction kit (EZ-10 Tatala RNA min-presps kit, shanghai) with reference to the kit instructions and a reverse transcription kit (PrimeScript) TM RT reagent Kit with gDNA Eraser, takara) into cDNA. Primers ds-F were designed based on the FAD gene sequence (sequence upload to NCBI database, PRJNA 489372): at (at)gagctcgcaatgatacaacgaacca (SEQ ID NO. 1) and ds-R: at (at)gtcgacgaacgatgtgaccataagc (SEQ ID NO. 2) the expected amplified fragment size was 266bp and PCR amplification was performed using cDNA as template [ Taq PCR Master Mix (2X, with Red Dye), division of Biotechnology (Shanghai)]The PCR reaction system is shown in Table 1. Reaction conditions: pre-denaturation at 95℃for 2min; denaturation at 95℃for 30s, annealing at 50℃for 30s, elongation at 72℃for 1min,35 cycles; extending at 72deg.C for 10min, and preserving at 4deg.C.
TABLE 1
The result of the electrophoresis detection of the amplified fragments showed between 250-500bp (FIG. 2A), which was consistent with the expectations. According to the procedure of vector construction in FIG. 1, the FAD gene gel recovery product and L4440 vector were digested with SacI and SalI (NEB (Beijing)) in a water bath at 37℃for 10min, the reaction system was as shown in Table 2, and the digested products were recovered, and after detection by agarose gel electrophoresis, the digested products were ligated overnight with T4 ligase to give FAD-L4440 recombinant plasmid.
TABLE 2
The FAD-L4440 recombinant plasmid is transferred into 50 mu L DH5 alpha competent cells after being connected, coated on an SOC solid culture medium containing ampicillin, cultured for 12 hours, and single colony is selected and cultured in the SOC liquid culture medium. The PCR amplification is carried out by using T7 single primer bacterial liquid, the reaction system is shown in table 3, and the PCR reaction conditions are as follows: pre-denaturation at 95℃for 2min; denaturation at 95℃for 30s, annealing at 50℃for 30s, elongation at 72℃for 1min,35 cycles; extending at 72deg.C for 10min, and preserving at 4deg.C; the expected target band was obtained (FIG. 2B) and sent to the company for sequencing, and the result showed that the FAD-L4440 recombinant plasmid was successfully constructed, as shown by the coincidence with the target fragment. The T7 single primer sequence is taatacgactcactatagg (SEQ ID NO. 3).
TABLE 3 Table 3
Inducible expression of 2 dsRNA
Transferring FAD-L4440 recombinant plasmid into HT115 competent cells, and performing amplification culture until bacterial liquid OD 600 At 0.4, IPTG was added to induce for 4 hours, and the cells were collected (FIG. 1). The total RNA was detected in the induced bacterial liquid, and the results are shown in FIG. 2C. Gel electrophoresis results show that RNA extracted from FAD-L4440-HT115 bacterial liquid after induction contains an RNA band of an item gene, and RNA extracted from FAD-L4440-HT115 bacterial liquid before induction does not contain the band, so that the FAD-L4440-HT115 specific band after induction can be judged to be a dsRNA band of FAD, and the successful induction expression of FAD gene dsRNA is proved.
Wherein the expansion culture specifically comprises the following steps: transferring the constructed FAD-L4440 recombinant plasmid into HT115 competent strain, coating on SOC solid culture medium containing ampicillin and tetracycline, culturing 12h, selecting single colony, transferring into SOC liquid culture medium containing ampicillin and tetracycline, culturing overnight at 37deg.C, adding the overnight culture solution into 2 XYT liquid culture medium containing ampicillin and tetracycline, and culturing to OD 600 =0.4。
3. Transfection effect detection
Diluting thallus with sterile water to obtain three different concentrations of test bacterial liquid (620 ng. ML) -1 、62 ng•mL -1 、6.2 ng•mL -1 ) As an experimental group, the natural group (ck) was not treated with any treatment, and the L4440-HT115 bacterial liquid (the preparation process was the same as that of the FAD-L4440-HT115 bacterial liquid, except that: recombinantPlasmid without FAD gene fragment) was used as a control group. The shellac is secreted in a large amount in the shellac larva stage to gradually form a rubber shell to cover the larva and the host plant branches, and the shellac is hardly absorbed by the larva after being coated and sprayed with dsRNA bacterial liquid, so that the shellac larva stage is selected to be transfected.
(1) And (5) coating: FAD-L4440-HT115 thalli are diluted into different concentrations by sterile water, and then are directly smeared on the bodies by a soft brush.
(2) Spraying: diluting FAD-L4440-HT115 thalli with sterile water to different concentrations, and directly spraying on the thalli by using a small spray can.
The experimental samples collected at 12h, 24h, 48h and 72 h after 3 consecutive days of treatment were treated 2 times per day. Fluorescent quantitative primers are designed according to FAD gene fragments, and upstream primers: catcgttcttacaaggctaa (SEQ ID NO. 4) and downstream primer: tatgtggatcggcattcg (SEQ ID NO. 5), and selecting β -actin as reference gene, upstream primer: atcgtgctgagtgaggaa (SEQ ID NO. 6) and downstream primer: cgcttcgctgattatcgta (SEQ ID NO. 7). The detection was performed using a fluorescent quantitative (RT-qPCR) kit (SYBR Primix Ex TaqTM, takara Co.) and the reaction system was as shown in Table 4, and the reaction conditions were: pre-denaturation at 95℃for 2min; denaturation at 95℃for 30s, annealing at 50℃for 30s, elongation at 72℃for 1min,35 cycles, 2 were used -∆∆CT The relative expression level was calculated by the method.
TABLE 4 Table 4
The results showed that the FAD gene expression level was significantly reduced in the group treated with the dried FAD-L4440-HT115 bacterial liquid, with a significant difference between the low and medium concentrations at 12h relative to the control group, which was reduced by 90.79% and 85.46%, respectively, and the interference efficiency at a medium concentration of 72 h was higher than in the other two groups (FIG. 3). The FAD gene expression level treated with the sprayed FAD-L4440-HT115 bacterial liquid was reduced at low concentrations of 12h and 48h and at medium concentrations of 24h and 48h, with only a significant difference at medium concentration of 48h relative to the control group (FIG. 4).
Collecting different treated shellfishes after one month, peeling fresh glue blocks, weighing, soaking the glue blocks with 95% alcohol, filtering, taking the number of individuals of the shellfishes, weighing, and calculating the individual glue yield. The result shows that compared with the low-concentration and medium-concentration treatment groups, the ck group individual gum secretion amount of the dry coating method has obvious differenceP< 0.05), reduced by 14.89% and 12.73%, respectively, and the gum yield of the ck group individuals was increased compared with that of the experimental groups treated with 3 different concentrations by spraying (figure 5).
By comparing the two treatment methods of coating and spraying, the coating method can effectively interfere the FAD gene expression quantity, thereby reducing the glue yield of individuals, but the interference effect of the spraying method is not obvious.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> national institute of forestry science research resource insect institute
<120> a method for mediating double-stranded RNA to enter Chinese purple worm body
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
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atgagctcgc aatgatacaa cgaacca 27
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<213> Artificial sequence ()
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atgtcgacga acgatgtgac cataagc 27
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<213> Artificial sequence ()
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taatacgact cactatagg 19
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catcgttctt acaaggctaa 20
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<212> DNA
<213> Artificial sequence ()
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tatgtggatc ggcattcg 18
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atcgtgctga gtgaggaa 18
<210> 7
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<212> DNA
<213> Artificial sequence ()
<400> 7
cgcttcgctg attatcgta 19
Claims (6)
1. A method for mediating double-stranded RNA to enter the body of rhodochrous sinensis, comprising the steps of:
step (1), extracting total RNA of the Chinese red beetles, carrying out reverse transcription to cDNA, and then carrying out PCR amplification, wherein an amplification product and an L4440 vector are subjected to double enzyme digestion by SacI and SalI, and the enzyme digestion product is connected overnight by using T4 ligase to obtain FAD-L4440 recombinant plasmid;
step (a)2) Transferring the FAD-L4440 recombinant plasmid obtained in the step (1) into HT115 competent cells, and performing amplification culture until bacterial liquid OD is obtained 600 Adding IPTG to induce for 2-5 h to obtain FAD-L4440-HT115 bacteria at 0.1-0.7;
step (3), diluting FAD-L4440-HT115 cells obtained in the step (2) to a concentration of 6.2ng -1 ~620ng•mL -1 Directly smearing the diluted bacterial liquid on the larva stage insect body of the Chinese red beetle;
the total RNA of the Chinese purple beetles is total RNA containing FAD genes of the Chinese purple beetles;
in the step (1), the primers adopted in the PCR amplification are ds-F and ds-R, wherein,
ds-F：atgagctcgcaatgatacaacgaacca；
ds-R：atgtcgacgaacgatgtgaccataagc。
2. the method of mediating double stranded RNA into the body of Chinese violet according to claim 1, wherein in step (1), the PCR amplification system is
Taq PCR Master Mix(2X, withRed Dye) 12.5μl，
cDNA 1.0μl，
ds-F 10μM 1.0μl，
ds-R 10μM 1.0μl，
dH2O 9.5μl，
And a total of 25.0 μl.
3. The method of mediating double stranded RNA into the body of chinese rhodopsin according to claim 2, wherein in step (1), the PCR reaction conditions are as follows: pre-denaturation at 95℃for 2min; denaturation at 95℃for 30s, annealing at 50℃for 30s, elongation at 72℃for 1min,35 cycles; extending at 72deg.C for 10min, and preserving at 4deg.C.
4. The method of mediating double stranded RNA into the body of chinese rhodopsin according to claim 1, wherein in step (1), the time of cleavage is 10min and the temperature is 37 ℃.
5. The method of mediating double stranded RNA into the body of chinese rhodopsin according to claim 1, wherein in step (1), the system of cleavage is:
SacⅠ 1.0 μl，
SalⅠ 1.0 μl，
Buffer 5.0 μl，
FAD or L4440.0. Mu.l,
dH 2 O 38.0 μl，
and total 50.0 μl.
6. The method for mediating double-stranded RNA into the body of Chinese purple worm according to claim 1, wherein in the step (2), the specific method for expanding culture is as follows: the constructed FAD-L4440 recombinant plasmid is transformed into HT115 competent strain, coated on SOC solid culture medium containing ampicillin and tetracycline, cultured for 12h, single colony is selected and transferred into SOC liquid culture medium containing 100 mug/mL ampicillin and 50 mug/mL tetracycline for overnight culture at 37 ℃, then the overnight culture solution is added into 2 XYT liquid culture medium containing 75 mug/mL ampicillin and 12.5 mug/mL tetracycline for culturing to OD 600 =0.1~0.7。
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| CN104404048A (en) * | 2012-11-28 | 2015-03-11 | 石河子大学 | Method for effectively controlling agricultural pest mites by RNAi |
| CN107278228A (en) * | 2014-12-16 | 2017-10-20 | 美国陶氏益农公司 | The parental generation RNAi of HUNCHBACK genes for controlling Hemipteran pest suppresses |
| CN107683088A (en) * | 2015-05-27 | 2018-02-09 | 美国陶氏益农公司 | Assign the THREAD nucleic acid to the patience of Hemipteran pest |
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| Molecular phylogeny and biogeography of lac insects (Hemiptera: Kerriidae) inferred from nuclear and mitochondrial gene sequences;Zixiang Yang et al.;《Molecular Biology Reports》;20130928;第5943–5952页 * |
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